S190: ASXL1 MUTATIONS ACCELERATE BONE MARROW FIBROSIS VIA EGR1-TNFA AXIS MEDIATED INFLAMMATION AND FIBROCYTE GENERATION IN MYELOPROLIFERATIVE NEOPLASMS
Authors: Z. Shi, J. Liu, Y. Zhao, L. Yang, Y. Cai, P. Zhang, Z. Xu, T. Qin, S. Qu, L. Pan, J. Wu, X. Yan, Z. Li, W. Zhang, Y. Yan, H. Huang, G. Huang, B. Li, X. Wu, Z. Xiao
Journal: HemaSphere (2022)

Background: Apart from the central role of activated JAK/STAT signaling pathway, ASXL1 mutations are the most recurrent additional mutations in myeloproliferative neoplasms (MPNs) and occurred much more common in myelofibrosis (MF) than essential thrombocythemia (ET) and polycythemia vera (PV) patients. However, the mechanism of the association with ASXL1 mutations and bone marrow (BM) fibrosis remains unknown. Aims: To investigate the role of ASXL1 mutations in MF acceleration and explore the underlying mechanism. Methods: We studied the clinical data of 302 consecutive MF patients in our hospital. For mouse model study, Vav1-Cre mice, Asxl1flox/flox and Jak2V617F/+ knockin alleles were used to achieve hematopoietic cell-specific Jak2V617F/+/Asxl1flox/flox (Asxl1-/-Jak2VF), Jak2V617F/+ (Jak2VF), and Asxl1flox/flox (Asxl1-/-) mice. Integrated analysis of RNAseq, transposase-accessible chromatin using sequencing (ATAC-Seq) and chromatin immunoprecipitation (ChIP)-seq were performed to explore the underlying mechanism. Results: 98 of 302 (32.5%) patients harbored ASXL1 mutations. ASXL1 mutations are correlated with higher monocyte counts, increasing CD34+ cells in peripheral blood (PB), larger spleen sizes, and higher MF grades. Consistent with clinical findings, Asxl1-/-Jak2VF mice showed lower platelet counts, higher monocyte counts and c-kit+ cells in PB compared with Jak2VF mice. Hematopoietic stem and progenitor cells (HSPCs) compartment analysis revealed increased granulocyte/macrophage progenitors (GMPs), and megakaryocyte /erythroid progenitors (MEPs) in spleens of Asxl1-/-Jak2VF, not in BM compared with Jak2V617F mice (Figure 1A), in line with increased spleen weights of Asxl1-/-Jak2VF mice (Figure 1B). Histology analysis revealed reticulin infiltration in BM of Asxl1-/-Jak2VF mice, not in other age-matched genotypes. Both ASXL1MT MF patients and Asxl1-/-Jak2VF mice showed increased monocytes/macrophages in BM (Figure 1C-D), accounting for more severe inflammation in ASXL1MT MF patients and mouse models. In vitro differentiation assay revealed monocyte/ macrophage differentiation bias of Asxl1-/-Jak2VF HSPCs (Figure 1E). In fibrosis driving cells analysis, ASXL1MT MF patients showed comparable mesenchymal stromal cells (MSCs) derived myofibroblasts while increased monocyte derived fibrocytes in BM compared with ASXL1WT MF patients (Figure 1F). In vitro fibrocyte differentiation assay (Figure 1G) and flow cytometry analysis confirmed increased fibrocytes in BM of Asxl1-/-Jak2VF mice compared with other genotypes. Mechanismly, Asxl1 deletion in Jak2VF HSPCs leads to enhancer activation of polycomb group (PcG) target genes, such as Egr1(Figure 1H). The upregulation of Egr1, which was validated in mouse models and patients (Figure 1I-J), in turn accounts for increased HSPCs commitment to monocyte/macrophage lineage (Figure 1K). Moreover, Egr1 induces the activation of Tnfa(Figure 1L) and thereby further drives the differentiation of monocytes to fibrocytes (Figure 1M). Accordingly, combined TNFR antagonist (R-7050) with ruxolitinib significantly reduces fibrocytes production in vitro in Asxl1-/-Jak2VF mice and ASXL1MT MF patients (Figure 1N-O). Image:Summary/Conclusion: Our study illustrated the crucial role of ASXL1 mutation in MPN phenotypes and BM fibrosis. ASXL1 mutations activate the EGR1-TNFA axis in MPNs, leading to monocyte/macrophage mediated inflammation and fibrocyte induced BM fibrosis. Ruxolitinib along with TNFR antagonist may ameliorate fibrocytes, providing an attractive theoretical approach for antifibrosis treatment.